title: Lyso-myristoyl phosphatidylcholine micelles sustain the activity of Dengue non-structural (NS) protein 3 protease domain fused with the full-length NS2B.
abstract: Dengue virus (DENV), a member of the flavivirus genus, affects 50-100 million people in tropical and sub-tropical regions. The DENV protease domain is located at the N-terminus of the NS3 protease and requires for its enzymatic activity a hydrophilic segment of the NS2B that acts as a cofactor. The protease is an important antiviral drug target because it plays a crucial role in virus replication by cleaving the genome-coded polypeptide into mature functional proteins. Currently, there are no drugs to inhibit DENV protease activity. Most structural and functional studies have been conducted using protein constructs containing the NS3 protease domain connected to a soluble segment of the NS2B membrane protein via a nine-residue linker. For in vitro structural and functional studies, it would be useful to produce a natural form of the DENV protease containing the NS3 protease domain and the full-length NS2B protein. Herein, we describe the expression and purification of a natural form of DENV protease (NS2BFL-NS3pro) containing the full-length NS2B protein and the protease domain of NS3 (NS3pro). The protease was expressed and purified in detergent micelles necessary for its folding. Our results show that this purified protein was active in detergent micelles such as lyso-myristoyl phosphatidylcholine (LMPC). These findings should facilitate further structural and functional studies of the protease and will facilitate drug discovery targeting DENV.
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A∗STAR), Singapore 138669, Singapore.
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Huang
authors.forename: Qingxin
authors.initials: Q
authors.lastname: Li
authors.forename: Joma
authors.initials: J
authors.lastname: Joy
authors.forename: Angela Shuyi
authors.initials: AS
authors.lastname: Chen
authors.forename: David
authors.initials: D
authors.lastname: Ruiz-Carrillo
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Hill
authors.forename: Julien
authors.initials: J
authors.lastname: Lescar
authors.forename: Congbao
authors.initials: C
authors.lastname: Kang
mesh_terms: D000595:Amino Acid Sequence; D003902:Detergents / Q000737:chemistry; D004795:Enzyme Stability; D008244:Lysophosphatidylcholines / Q000737:chemistry / Q000494:pharmacology*; D008823:Micelles; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D003902:Detergents; D008244:Lysophosphatidylcholines; D008823:Micelles; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C035255:myristoyllysophosphatidylcholine
keywords: Dengue virus; Detergent micelles; Membrane protein; NS2B; Protease